Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin

ABSTRACT Gram-negative bacteria are problematic for antibiotic development due to the low permeability of their cell envelopes. To rationally design new antibiotics capable of breaching this barrier, more information is required about the specific components of the cell envelope that prevent the passage of compounds with different physiochemical properties. Ampicillin and benzylpenicillin are β-lactam antibiotics with identical chemical structures except for a clever synthetic addition of a primary amine group in ampicillin, which promotes its accumulation in Gram-negatives. Previous work showed that ampicillin is better able to pass through the outer membrane porin OmpF in Escherichia coli compared to benzylpenicillin. It is not known, however, how the primary amine may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect E. coli fitness in the presence of equivalent subinhibitory concentrations of ampicillin and benzylpenicillin, with a focus on the cell envelope. Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had less effect on fitness under ampicillin treatment. These results align with expectations if benzylpenicillin is poorly able to pass through porins. Disruption of genes encoding the AcrAB-TolC efflux system were detrimental to survival under both antibiotics, but particularly ampicillin. Indeed, insertions in these genes and regulators of acrAB-tolC expression were differentially selected under ampicillin treatment to a greater extent than insertions in ompF. These results suggest that maintaining ampicillin efflux may be more significant to E. coli survival than full inhibition of OmpF-mediated uptake. IMPORTANCE Due to the growing antibiotic resistance crisis, there is a critical need to develop new antibiotics, particularly compounds capable of targeting high-priority antibiotic-resistant Gram-negative pathogens. In order to develop new compounds capable of overcoming resistance a greater understanding of how Gram-negative bacteria are able to prevent the uptake and accumulation of many antibiotics is required. This study used a novel genome wide approach to investigate the significance of a primary amine group as a chemical feature that promotes the uptake and accumulation of compounds in the Gram-negative model organism Escherichia coli. The results support previous biochemical observations that the primary amine promotes passage through the outer membrane porin OmpF, but also highlight active efflux as a major resistance factor.

In the introduction the authors state that this study investigated the importance of the primary amine group in promoting uptake. I think the final conclusions could be strengthened by returning to this posed question and commenting on what the data tells us about the impact of the amine.

Reviewer #2 (Comments for the Author):
In this study the authors used TraDIS to identify genes that affect E. coli fitness in the presence of sub-inhibitory concentrations of ampicillin or benzylpenicillin. It is important to understand the mechanisms that cause resistance to antibiotics in order to design methods to overcome these resistance mechanisms. This study is therefore timely and important.
In general, mutations in the outer membrane, particularly the lipopolysaccharide layer decreased the fitness under benzylpenicillin exposure. This result is in line with the notion of benzylpenicillin not being an effective antibiotic against Gramnegative organisms due to its inability to pass through outer membrane porins such as OmpF.
Some of the other findings could have been explored a bit more for example the effect of mutations in dacA.
Line 176 and onwards: The argument about the PEN being transported at a higher rate compared to AMP and the relationship between the inner membrane permeability and mutations in the regulatory machinery of AcrAB-TolC, needs to be better explained.
There is a few minor corrections needed for example: Line 19 -Should this sentence read "...components of the envelope that prevent....." Line 81 -E. coli not E.coli Staff Comments:

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Reviewer comments:
Reviewer #1 (Comments for the Author): This interesting study by Maher et al., describes a TraDIS approach to investigating the effect of two structurally very similar antibiotics ampicillin and benzylpenicillin. The use of an unbiased screen to address how these compounds accumulate in E. coli is a strength of this study and has produced interesting results which will be of interest to the field. That said it is a description of a single experiment (with validation) so I think fits well into the observation format.
I appreciate that due to the article format there is no actual methods section but I think more methodological detail is required. For example, what growth phase were the cells grown to as this may well affect the genes that were detected.
Authors' response: Additional detail about library challenge methodology was added to the revised manuscript. Lines 87-89 "Cells were sampled at stationary phase to ensure the effects of compound accumulation throughout the cell cycle, including during active growth phases, were reflected in mutant abundance (10)".
In the introduction the authors state that this study investigated the importance of the primary amine group in promoting uptake. I think the final conclusions could be strengthened by returning to this posed question and commenting on what the data tells us about the impact of the amine.